Development and Antibacterial Performance of Novel Polylactic Acid

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Development and Antibacterial Performance of Novel Polylactic Acid-Graphene Oxide-Silver Nanoparticle Hybrid Nanocomposite Mats Prepared By Electrospinning Chen Liu, Jie Shen, Kelvin W.K. Yeung, and Sie Chin Tjong ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00766 • Publication Date (Web): 16 Jan 2017 Downloaded from http://pubs.acs.org on January 18, 2017

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ACS Biomaterials Science & Engineering

Development and Antibacterial Performance of Novel Polylactic AcidGraphene Oxide-Silver Nanoparticle Hybrid Nanocomposite Mats Prepared By Electrospinning Chen Liu,‡,† Jie Shen,‡,§ Kelvin Wai Kwok Yeung,§ and Sie Chin Tjong,*, † †

Department of Physics and Materials Science, City University of Hong Kong, Kowloon,

Hong Kong §

Department of Orthopedics and Traumatology, Li Ka Shing Faculty of Medicine, The

University of Hong Kong, Hong Kong * E-mail address: [email protected] Antibacterial nanomaterials have attracted great interests in recent years, especially with an increase of antibiotic resistance of microbial organisms. However, deleterious properties such as aggregation, toxicity of nanoparticles and low stability limit their practical application. In this respect, we have developed novel PLA-based fibrous mats with GO-Ag hybrid nanofillers through electrospinning for minimizing bacterial attachment and growth for biomedical applications. Polylactic acid (PLA) exhibits low tensile modulus and strength as well as no bactericidal ability. To enhance its tensile and bactericidal performances, 1 wt% graphene oxide (GO) and 1-7 wt% silver nanoparticle (AgNP) are incorporated into the PLA matrix. For comparison, electrospun PLA-1wt% GO and PLA-AgNP nanocomposites have also been prepared. The morphological, mechanical and thermal properties as well as bactericidal activities of electrospun PLA-based nanocomposite fibrous mats have been investigated. Tensile tests show that the addition of 1wt% GO or 1-7 wt% AgNPs to PLA leads to a drastic increase in its elastic modulus. Further enhancements in tensile modulus and strength of PLA can be obtained by adding GO-AgNP nanohybrids. The thermal stability of PLA is greatly improved by adding GO-AgNP nanohybrids. Agar disk diffusion test results

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indicate that the PLA-1wt%GO nanocomposite has no inhibition zones against Esherichia coli (E. coli) and Staphylococcus aureus (S. aureus). However, GO nanofillers with lateral width of micrometer range act as effective anchoring sites for AgNPs. Thus, PLA-1wt%GO(1-7) wt% Ag hybrid fibrous mats exhibit excellent antibacterial effect against E. coli, while the PLA-1wt%GO-Ag mats with higher AgNP loadings show bacterial inhibition towards S. aureus. The bactericidal effects of PLA-1wt%GO-(1-7)%Ag hybrids are studied and analyzed using live/dead fluorescent imaging assay, quantitative antibacterial efficacy test, SEM examination and residual oxygen species measurement. Our work highlights the development of electrospun nanocomposite mats as promising antibacterial materials for biomedical applications and systematically depicts the bactericidal mechanism of PLA-GOAg nanocomposites. KEYWORDS: polylactic-acid; graphene oxide; silver nanoparticles; hybrid nanocomposite; electrospinning; antibacterial activity 1. Introduction Over the past decades, the worldwide increase of antibiotic resistance of bacteria has been a severe threat to public health. Microorganisms commonly attach to the surfaces of medical supplies devices and implant materials to form biofilms, which are surrounded by an extracellular polymeric matrix of exopolysaccharide (EPS).1,2 Poor disinfection practices and ineffective cleaning products may increase the infection risk associated with pathogenic organisms. Recent advances in nanotechnology have provided new insights into antimicrobial agents, and led to the development of functional nanomaterials with unique chemical and physical properties. Antibacterial agents such as silver, copper, zinc and zinc oxide nanoparticles can reduce the attachment and viability of microbes on biomedical supplies surfaces.3-7


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Silver nanoparticles (AgNPs) with high surface area to volume ratio exhibit inhibitory and bactericidal effects.8-11 AgNPs have the ability to interact with bacteria through which they destroy the membrane, cross the body of the microbe and create intracellular damage.12 Moreover, graphene and its derivatives are promising candidates in biotechnology and nanomedicine for developing biosensors for the detection of biologically active molecules.13,14 Graphene sheets can be prepared by means of mechanical exfoliation of graphite using the so-called ‘scotch-tape’ technique, chemical exfoliation of graphite in a solvent under sonication and chemical vapor deposition (CVD). Both mechanical and chemical exfoliation of graphite can produce high purity graphene, but their production yield is very low especially the former. Mechanically exfoliated graphene is typically used for scientific research purposes for investigating its chemical, physical and mechanical properties. CVD-growth graphene sheets are usually employed in the electronic and optoelectronic sectors for forming transparent conducting electrodes of solar cells and computer touch screen panels.15 However, bulk quantities of graphene can be prepared through chemical oxidation of graphite flakes in strong oxidizing solutions to generate graphene oxide (GO), followed by either chemical or thermal reduction treatment to form reduced GO. Two-dimensional GO sheets bear several oxygen-containing surface groups, including hydroxyl, epoxide, carbonyl and carboxyl groups. In contrast to conventional polymer microcomposites having large filler contents,16-19 GO sheets act as an effective nanofiller for polymers at low loading levels.20-25 These functional groups can improve interfacial bonding between the polymeric matrix and GO, leading to efficient stress transfer across the GO-polymer matrix of polymer nanocomposites during mechanical tests. This is a typical characteristic of polymer biocomposites reinforced with carbonaceous nanofillers.26 In particular, GO additions are beneficial in enhancing biocompatibility of degradable polymers.22,27-30 As an example, Pinto et al. reported that GO nanofillers enhance the



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adhesion and proliferation of fibroblasts on the surface of GO/PLA film.23 The hydroxyl and carboxyl groups of GO sheets increase hydrophilicity of the PLA film, thereby enhancing cell-material interactions. Silver-based antimicrobial materials are particular attractive because silver has strong biocidal effects on numerous bacteria species provided by its ions. The development of novel AgNP-GO hybrid materials has attracted considerable research interest recently for various applications in catalysis,31,32 osmosis membrane33,34 and bactericidal activity.35-39 The hybrid materials exhibit synergistic antibacterial properties that exceed the performances of individual GO and AgNPs components. Electrospinning is a simple and versatile process capable of producing continuous polymer fibers with diameters in the micron to nanoscale range onto a target by regulating the ejection of polymeric fluid jet from the syringe.40

Polymer nanofibrous mats prepared by

electrospinning have high porosities with interconnected pores, and exhibit morphological similarities to the natural extracellular matrix (ECM).41-44 The fibrous component of ECM in the tissue consists of protein fibers such as collagens, elastin, laminin, fibronectin and vitronectin. These protein fibers provide structural support to tissues and regulate many aspects of cell behavior.41 In this respect, electrospun nanofibers find attractive applications in biomedical engineering including tissue-engineering scaffolds, controlled drug release depots, wound dressings, coatings for medical-implant devices, nanocomposite materials for dental restoration, membranes for molecular separations, and biosensor coatings.43 Synthetic polymers based on aliphatic polyesters, such as polylactic acid (PLA), polyglycolide (PGA) and their copolymers have been widely used for biomedical applications.45-49 These polymers undergo degradation through hydrolysis of the ester groups in their backbones. The degradation rates and products can be tuned according to the composition, structure, and molecular weight. In general, PGA degrades rapidly in aqueous


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solutions and loses its mechanical strength between two and four weeks because of its relatively hydrophilic nature. By contrast, PLA is more hydrophobic than PGA, due to the presence of extra methyl group in its repeating units. It usually requires several months to years for PLA to lose mechanical integrity in vitro or in vivo.50 Recently, Kurtycz et al. fabricated electrospun PLA-Al2O3-Ag composite fibers for antibacterial purposes.51 Zhu et al. prepared PLA-hydroxyapatite-Ag nanocomposites with bactericidal properties for biomedical applications.52 From the literature, GO has been incorporated into biodegradable polymers to produce fibrous scaffolds during electrospinning.29,30,53-55 As mentioned, hybridization of GO with AgNPs renders the composite materials with bactericidal capability. In this respect, Liu and coworkers prepared GO-AgNPs@polyethylene glycol (PEG) bulk composites by reducing AgNO3 with sodium citrate or NaBH4 in the presence of GO@PEG suspension. The resulting GO-AgNPs@PEG polymer composites exhibited markedly higher antibacterial efficacy than AgNPs alone.56 However, little information is available on the formation of electrospun polymer nanocomposites with GO-AgNPs hybrid fillers. Very recently, Li et al. prepared nanofibrous membrane electrodes consisting of polyvinyl alcohol/GO-AgNPs for electrochemical sensing purposes.57 Their work involved the material characterization of resulting hybrid electrodes, but no tests were performed on antimicrobial resistance. De Faria et al. fabricated electrospun mats by adding GO-AgNPs fillers to poly(lactide-co-glycolide) (PLGA) and chitosan blend for developing flexible coating materials.58 The development of antibacterial polymer nanocomposites is critical for clinical application of biomedical supplies for preventing infection. Hospital-acquired infections associated with the use of invasive medical devices or surgical procedures are a major challenge to patient safety.59 It is the purpose of this paper to form antibacterial mats by incorporating AgNPs-GO hybrids into PLA for clinical applications through electrospinning. It is the first study of electrospun PLA-



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GO-AgNP hybrid fibrous mats illustrating such a detailed and systematic investigation of their bactericidal activities. 2. Experimental 2.1. Materials and Reagents PLA pellets were purchased from Shenzhen Bright China Inc. Graphite flakes were bought from Sigma-Aldrich Inc. (St. Louis, MO, USA). All reagents such as silver nitrate (AgNO3), sulfuric acid (H2SO4), hydrogen peroxide (H2O2), potassium permanganate (K2MnO4), sodium nitrate (NaNO3), N, N-dimethylformamide (DMF), dichloromethane (DCM), ethanol, formalin and phosphate-buffered saline (PBS) were of analytical grade and used as received. Lysogeny broth (LB) and brain heart infusion (BHI) broth were purchased from Thermo Fisher Scientific Inc. (USA). 2.2. Synthesis of GO-Ag nanohybrids GO was first synthesised using a modified Hummers’ method. In brief, graphite flakes (2.0 g) were oxidized chemically in a 1000 mL volumetric flask containing concentrated H2SO4 (68 mL) with NaNO3 (1.9 g), and the mixed solution was then cooled in an ice bath under continuous stirring. Later, K2MnO4 (9.0 g) was added gradually to the solution mixture, and stirred for 2 days. This was then diluted with slow addition of 100 mL water. Finally, the solution was treated with 2.5 mL H2O2 to terminate the reaction. The resulting solution was centrifuged and the supernatant was decanted away. The remaining substance was rinsed with 3% HCl and water respectively followed by freeze-drying. For preparing the GO-Ag nanohybrids, GO was dispersed in DMF for 30 min under sonication, followed by adding precise amounts of silver precursor, i.e. AgNO3. Here DMF acted as a reducing agent and reaction medium.60 The suspension was further sonicated for 30


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min to form a stable brown colloid solution. Subsequently, the reaction was magnetic stirred for 10 h at 60 °C until AgNO3 was reduced to AgNPs completely.61 For comparison, AgNPs were obtained by directly adding AgNO3 to DMF without GO addition and followed by the same synthesis process of GO-Ag nanocomposites. 2.3. Fabrication of electrospun fibrous mats To prepare electrospun PLA fibrous mat, PLA pellets were dissolved in a mixture of DCM and DMF (75:25, v/v) directly. The homogenized solution was obtained by stirring overnight at room temperature. For preparing PLA-Ag and PLA-GO-Ag nanocomposite fibers, the appropriate weight of PLA was dissolved in DCM and stirred until a transparent colloid solution was obtained, followed by adding desired amounts of Ag or GO-Ag dispersed in DMF. The mixed solutions were homogenized by magnetic stirring for another 1 h. Pure PLA and its composite fibrous mats were made from a nanofiber electrospinning unit (NEU; Kato Tech Co., Japan). For making PLA-GO-Ag hybrid fibers, the GO content was fixed at 1 wt% while the AgNPs contents were maintained at 1, 3, 7 wt% relative to the weight of PLA. In the process, working solution was pumped into a stainless steel needle tip with an orifice diameter of 0.9 mm from a syringe system. The mixture was ejected from the stainless steel needle with a voltage of 15-18 kV at an ejection rate of 1 mL/h. The distance between the needle and the collector was maintained at 12 cm. Each Fibrous mat was collected on an aluminium foil wrapped around a grounded metallic mandrel, rotating at a speed of 3 m/min. The electrospun fibrous mats were then dried overnight in a vacuum dryer at 60 °C to remove the solvent residue. Scheme 1 is the schematic representation showing the strategy for making GO-Ag nanohybrids and PLA-GO-Ag nanocomposite fibrous mats.



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Scheme 1. Schematic illustration of the preparation of GO-Ag and electrospun PLA-GO-Ag nanocomposite fibrous mats. 2.4. Material characterization Graphene oxide and GO-AgNPs nanohybrids were first examined in an atomic force microscope (AFM; Veeco Nanoscope V). Transmission electron microscopy (TEM; Philips CM 20) and scanning electron microscopy (SEM; Jeol JSM-820) were employed to characterize electrospun PLA-GO and PLA-GO-Ag nanocomposite fibrous mats. The diameter of PLA-based fibers was determined from the SEM images by image analysis using ImageJ software (ImageJ, Bethesda, MD, USA). Thermogravimetric measurements were performed with the TGA1 STARe system (Mettler Toledo AG, Switzerland) from 50 to 480 °C in nitrogen atmosphere at a heating rate of 10°C/min. The temperatures at 10% weight loss (T10%) and 20% weight loss (T20%) were obtained from the weight loss vs temperature curves. The tensile properties of electrospun fibrous mats were measured with an Instron tester (model 5567) under a crosshead speed of 10 mm/min at room temperature. All fibrous mats were 8 ACS Paragon Plus Environment

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cut into rectangular specimens of 50 mm (length) and 10 mm (width) with a gauge length of 30 mm. The stress-strain curves of fibrous mats were measured accordingly. Five samples of each composition were tested and the average value was reported. 2.5. Silver ion release and contact angle tests Electrospun PLA-Ag and PLA-GO-Ag nanocomposite fibrous mats were cut into round pieces with a diameter of 10 mm and then immersed in 10 mL deionized water for different periods from 1 to 28 days. The Ag+ concentration released from the silver containing fibrous mats was quantitatively measured with inductively coupled plasma atomic emission spectrometry (ICPAES; Perkin Elmer 3300DV). Water contact angle measurements on PLA and its nanocomposite fibrous mats were conducted using Rame Hart 500-F1 advanced goniometer (Rame-Hart Instrument Co., NJ, USA) to reveal their surface wetting characteristics. The tests were performed using deionized water drops as the probe liquid on the specimen surfaces at room temperature. Six measurements were averaged to obtain contact angle for each material sample. 2.6. Antibacterial activity tests Both Gram-negative Escherichia coli (E. coli, ATCC 25922) and Gram-positive Staphylococcus aureus (S. aureus, ATCC 29213) were employed for different antibacterial assays. E. coli and S. aureus were grown in fresh lysogeny broth (LB) and brain heart infusion (BHI) broth respectively, and incubated in a shaking incubator at 37 °C overnight. The two bacterial suspensions were adjusted to reach the desired bacterial concentration by McFarland method. 9 ACS Paragon Plus Environment


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The antibacterial activities of samples studied were evaluated by Bauer-Kirby disk diffusion method.62 Bacterial suspension of 1 × 105 CFU/mL was evenly spread over the surface of the solid agar plates, followed by placing sterilized disk specimens (6 mm diameter) onto them. Following incubation agar plates at 37 °C for 18 h, bactericidal efficiency of each specimen against E. coli and S. aureus was assessed and compared by measuring the diameter of inhibition zone with a metric ruler having millimeter calibration. Six samples of each electrospun nanocomposite were used for the tests. Silver nitrate solution was dropped to a sterile filter paper (6 mm diameter) and used as positive control. Silver nitrate tended to release Ag+ ions for bactericidal activity.63 2.7. Quantitative analysis of antibacterial efficacy Samples were cut into disk specimens (10 mm diameter), soaked in the test tubes containing respective nutrient broth for E. coli and S. aureus of 1 × 106 CFU/mL, and then placed in a rotary shaker at 37 °C. The optical density at 600 nm (OD 600) of bacterial culture medium was recorded spectrophotometrically over time at an interval of 2 h up to 6h to monitor the growth rate of bacteria. The reduction of bacteria was calculated using the following equation: Bacterial reduction (%) = 100 × (B-A)/B

(1)

where A was the OD 600 value of bacterial culture medium inoculated with the sample, and B the OD 600 value of bacterial culture medium without the sample. 0% implied no antibacterial efficacy. 2.8. Fluorescent-based cell wall/membrane integrity assay

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All samples were exposed to respective E. coli and S. aureus bacteria of 1 × 106 CFU/mL for 12 h at 37 °C, then rinsed three times with PBS, stained with live/dead fluorescent staining kit (L7012, Molecular Probes; Eugene, OR, USA) at room temperature for 15 min in dark. This kit contains SYTO 9, a green fluorescent DNA stain for all kinds of cells, and a red fluorescent DNA stain (propidium iodide) for cells with a compromised membrane. A fluorescent microscope (Nikon, Eclipse 80i) was employed to observe the images. Bacteria cells with intact membranes were stained green, whereas cells with compromised membranes were considered to be dead or dying were stained red. 2.9. Morphological observation of bacteria All samples were treated with E. coli and S. aureus bacteria of 1 × 106 CFU/mL respectively for 12 h at 37 °C. The samples were then collected, washed twice with PBS, followed by fixing with 10% formalin solution for 4 h and dehydrated in a graded series of ethanol solutions (30%, 50%, 70%, 90% and 100%) for 10 min. The morphologies of the attached bacteria on the sample surfaces were observed using a Jeol SEM after sputter coating with a thin gold film. 2.10. ROS generation test The generation of reactive oxygen species (ROS) reflects the level of oxidative stress in cells, which is an important indicator of cell death. The ROS levels generated in E. coli and S. aureus after treating with PLA-Ag and PLA-GO-Ag fibrous mats were measured with an oxidationsensitive fluorescent probe 2,7-dichlorofluorescein diacetate (DCFH-DA, Sigma-Aldrich). Bacteria with 1 × 106 CFU/mL was collected by centrifugation, washed twice with PBS and then incubated with 20 µM DCFH-DA in PBS for 30 min at 37 °C. Afterwards, the bacteria cells were washed twice to remove DCFH outside the cell. The cleaned cells were then exposed to 11 ACS Paragon Plus Environment


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PLA, PLA-Ag and PLA-GO-Ag fibrous mats for 18 h. The ROS level was examined by a microplate reader (BMG Polarstar Optima) at an excitation wavelength of 488 nm and an emission wavelength of 535 nm. The relative ROS level was evaluated by comparing the absorbance of bacteria incubated with PLA-Ag and PLA-GO-Ag samples with that of bacteria cells cultured with pure PLA. The tests were performed three times with five samples for each test run. 2.11. Statistical analysis Antibacterial experiments were repeated at least three times. All data were analyzed and expressed as means ± standard deviations. A one-way ANOVA was used to determine the significance level of difference and p < 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. GO-Ag nanohybrids Figure 1a shows the AFM image of GO deposited on a silicon substrate along with its height profile. Apparently, GO layer exhibits a thickness of 1 nm, being larger than the thickness for of pure graphene (0.335 nm).64 This is due to the presence of oxygenated groups in GO. The AFM and TEM images of GO-3%Ag hybrid reveal that the as-formed silver nanoparticles are homogeneously dispersed throughout GO sheet (Figs. 1b-d). In other words, Ag particles are successfully attached, anchored and evenly dispersed onto the GO sheet. AgNPs exhibit spherical morphology, with sizes ranging from about 20-50 nm (average size: 30.05 nm). Furthermore, GO of large surface area shows folding feature under TEM observation.65


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Figure 1.

AFM images of (a) GO and (b) GO-3%Ag nanohybrid (c) Low and (d) High

magnified TEM images of GO-3%Ag nanohybrid.



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Figures 2a shows FTIR spectra of GO and GO-(1-7) %Ag nanohybrids. The GO spectrum displays several bands associated with the vibration modes of various oxygenated groups on its surface. The oxygenated moieties induce C=O stretching vibrations from carboxylic and carbonyl groups at 1735 cm-1, C=C skeletal vibration from unoxidized graphitic domain at 1625 cm-1, C-OH stretching vibration of COOH group at 1411 cm-1, C-O-C breathing vibration of epoxy group at 1225 cm-1 and C-O stretching at 1052 cm-1.66 Furthermore, a broad band at about 3450-3700 cm-1 is related to the O-H stretching vibrations of the C-OH group and adsorbed water molecules. For 1%GO-Ag nanohybrids, a decrease in the intensity of absorption bands associated with the oxygenated functional groups can be observed, especially for broad hydroxyl band at ~ 3450-3700 cm-1 of nanohybrids of higher Ag loadings.

This implies that the

oxygenated groups of GO such as carboxylic acid, hydroxyl, or epoxide groups act as reduction centers for silver ions to form AgNPs.67,68 In this respect, GO is converted to reduced graphene oxide accordingly.69 Fig. 2b shows the FTIR spectra of representative PLA-1%GO-1%Ag and PLA-1%GO-3%Ag nanocomposite fibers. The spectrum of pure PLA is also shown for comparison purposes. Pure PLA spectrum shows a main C=O vibration peak at 1752 cm-1, CH3 asymmetrical scissoring at 1454 cm-1, C-O asymmetrical stretching and CH3 twisting at 1180 cm1

, C-O-C stretching at 1088 cm-1, C–CH3 stretching at 1045 cm-1 and C–COO stretching at 868

cm-1.70,71 Apparently, PLA bands can be readily seen in the spectra of PLA-1%GO-1%Ag and PLA-1%GO-3%Ag composite fibers. The 1044 cm-1 peak in the nanocomposite fibers is ascribed to the overlap of C–CH3 stretching of PLA at 1045 cm-1 and C-O stretching of GO at 1052 cm-1. In addition, the 1735 cm-1 band of GO overlaps with the 1752 cm-1 peak of PLA.


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Figure 2. FTIR spectra of (a) GO and GO-(1-7) %Ag specimens; (b) PLA and PLA-1%GO1%Ag and PLA-1%GO-3%Ag composite samples. 3.2. Fiber morphology and structure Figures 3a-d are representative SEM micrographs displaying the morphologies of pure PLA, PLA-1%GO, PLA-3%Ag and PLA-1%GO-3%Ag fibrous mats. Pure PLA fibers show smooth surface feature polymer concentration and the change of solution viscosity as a result of nanofiller additions.72,73 PLA has an average fiber diameter of 751 ± 103 nm as determined by the Image J software (inset of Fig. 3a). By contrast, the surface of PLA-1%GO nanocomposite fibers is rougher than that of pure PLA, and the mean diameter of fibers is 406 ± 13 nm (Fig. 3b). Similarly, the additions of 1, 3 and 7% Ag to PLA also lead to a reduction of mean fiber diameters to 412 ± 36 nm, 379 ± 27 nm and 217 ± 11 nm, respectively. The typical surface feature of the PlA-3% Ag fibers is shown in Fig. 3c. By hybridizing 1, 3 and 7% Ag to the PLA1%GO, the diameters of composite fibers become even finer, i.e. 397 ± 17 nm, 330 ± 25 nm and 214 ± 10 nm, respectively (Fig. 3d). Generally, the resultant fiber size and morphology depend on the material properties such as solution conductivity, viscosity, type of polymer, polymer concentration, etc., and operational conditions (e.g. applied voltage, nozzle size and distance to 15 ACS Paragon Plus Environment


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the collector).40 Increasing the solution conductivity or charge density can lead to more uniform fibers with smaller fiber diameter. Increasing polymeric solution conductivity cause more electric charges carried by the electrospinning jet. This causes higher elongation forces imposing to the jet under the electrical field.74,75As expected, AgNP nanofillers with high electrical conductivity enhance the conductivity of polymer solution, thus producing electrospun AgNPscomposite nanofibers with finer diameter than polymer fibers without AgNPs.76,77 Similarly, conducting reduced graphene oxide (RGO) fillers also increases the conductivity of polymer solution for electrospinning. Thus RGO offers an effective strategy to improve the uniformity and decrease the fibrous diameter simultaneously.78 As aforementioned, functional groups of GO such as carboxylic acid, hydroxyl, or epoxide groups act as reduction centers for silver ions to form AgNPs, thereby converting GO to RGO.67,68 From these, it appears that the reduction of composite fiber diameter can be attributed to the enhancement of solution conductivity due to the use of conductive fillers. As a result, the diameters of the composite fibers depend on the conducting filler contents employed under the same operational conditions (i.e. applied voltage, nozzle size and distance to the collector) for all samples. Therefore, the repeatability can be maintained accordingly at a given filler content. Figures S1a-c (Supporting Information; SI) show the TEM images of PLA, PLA-1%GO1%Ag, PLA-1%GO-3%Ag nanocomposite fibers. AgNPs can be seen and dispersed uniformly in the PLA-1%GO-1%Ag nanocomposite hybrid fibers. This is due to two-dimensional GO with large surface areas can act as anchoring sites for AgNPs during fiber processing. By increasing AgNP content in the hybrid fibers to 3 wt%, GO-AgNPs also dispersed evenly in the polymer matrix. Fig. 4 shows representative X-ray diffraction pattern of pure PLA, PLA-1%GO-3%Ag and PLA-1%GO-7%Ag nanocomposites. The patterns of nanocomposites reveal the presence of 16 ACS Paragon Plus Environment

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PLA peak at 16.8°, corresponding to the (110) reflection. In addition, several peaks locate at 38.3°, 44.5° and 64.7° can be attributed to the (111), (200) and (220) reflections of metallic Ag according to Joint Committee on Powder Diffraction Standards (JCPDS) no. 04-0783. These results demonstrate that AgNPs have been successfully incorporate into the PLA matrix.

Figure 3. SEM micrographs of electrospun (a) PLA, (b) PLA-1% GO, (c) PLA-3%Ag and (d) PLA-1% GO-3%Ag fibrous mats.



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Figure 4. XRD patterns of representative electrospun samples of PLA, PLA-1%GO-3%Ag and PLA-1%GO-7%Ag. 3.3. Tensile behavior Figure S2 (SI) shows the stress-strain curves of PLA and its nanocomposite fibrous mats. Table 1 lists the Young’s modulus and tensile strength of these samples determined from the tensile tests. Electrospun PLA fibers exhibit low elastic modulus and tensile strength as expected. All composite nanofibers have higher elastic modulus and tensile strength than PLA fibers. The elastic modulus and tensile strength of electrospun PLA fibers can be enhanced greatly by adding 1% GO only, especially the former property. This implies that GO nanofillers can carry the applied load during tensile testing owing to effective stress-transfer effect from the polymer matrix to the GO fillers. As recognized, graphene sheet exhibits exceptionally high elastic modulus of 1 TPa and tensile strength of 130 GPa.79 The presence of oxygenated groups in GO reduces its elastic modulus to 380-470 GPa or even lower, and the tensile strength to 87.9 MPa.80-82 The elastic modulus and tensile strength of GO are much higher than those of PLA 18 ACS Paragon Plus Environment

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with a modulus of 3.5 GPa and tensile strength of 48 MPa.83 Apparently, GO serves as effective reinforcing fillers for pure PLA at the expense of tensile strain or ductility. It is worth mentioning that the presence of porosity in electrospun PLA lowers the modulus from 3.5 GPa to 8.70 MPa, and the tensile strength from 48 MPa to 0.76 MPa. Similarly, the incorporation of 17% AgNPs into the PLA matrix also enhances its elastic modulus and tensile strength markedly. Such improvements in the stiffness and strength increase with increasing AgNP content (Table 1). Thus AgNPs act as reinforcing fillers in the composites. A reverse trend is observed in tensile strain of the composites as the AgNP content increases. This is due to the rigid nature of AgNPs formed in situ in the PLA matrix. Comparing with the PLA-(1-7%)Ag nanocomposites, hybridization of 1% GO with AgNPs further enhances both the stiffness and strength of PLA fibrous mat (Table 1). For example, the stiffness and strength of PLA-1%Ag increase from 269.59 MPa and 1.67 to 377.09 MPa and 2.06 MPa, respectively by adding 1% GO. These correspond to 39.87% and 23.35% increments in stiffness and strength. The PLA-1%GO-7%Ag nanocomposite exhibits the highest tensile stiffness and strength of 1211.05 MPa and 5.46 MPa. Apparently, the deposition of AgNPs on the GO surface exhibits a beneficial effect in enhancing the mechanical performance of PLA fibrous mat. Table 1. Tensile properties of electrospun PLA and its nanocomposite fibrous mats. Specimen

Elastic modulus (MPa)

Tensile strength (MPa)

PLA

8.70±0.90

0.76±0.04

PLA-1%GO

147.78±3.46

1.22±0.12

PLA-1%Ag

269.59±6.51

1.67±0.12

PLA-1%GO-1%Ag

377.09±5.33

2.06±0.20

PLA-3%Ag

412.23±8.54

3.06±0.54

PLA-1%GO-3%Ag

755.4±8.80

3.71±0.48

PLA-7%Ag

968.05±12.71

4.65±0.76

PLA-1%GO-7%Ag

1211.05±13.53

5.46±0.81



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3.4. Thermogravimetric analysis Thermogravimetric analysis (TGA) is a powerful tool for assessing thermal stability of polymers and their nanocomposites. The weight loss and derivative weight loss of PLA and its nanocomposte fibrous mats with respect to temperature are depicted in SI, Figs. S3a and S3b, respectively. The temperatures correspond to T10% (temperature for 10% weight loss) and T20% (temperature for 20% weight loss) of all samples studies are listed in Table 2. The T10% and T20% values of PLA increase from 330.66 ˚C and 340.99 ˚C to 341.53 ˚C and 350.31 ˚C, respectively by adding 1 wt% GO. It is obvious that GO nanofillers enhance the thermal stability of PLA by delaying the rate of thermal degradation. Thus GO nanofillers of large surface areas act as effective barriers for volatile constituents from diffusion into the PLA surface. By contrast, the addition of 1% AgNPs nanoparticles to PLA also leads to a slight increase in its T10% and T20% values to 332.47 ˚C and 342.99 ˚C, respectively. An improvement in thermal stability of PLA is also observed by adding 3% AgNPs, which is due to the more heat-stable metallic silver nanoparticles. A similar trend is observed for the maximum weight loss (Tmax) determined from derivative thermographic (DTG) curves of PLA-Ag and PLA-1%GO-Ag nanocomposite fibrous mats (Table 2). This Table also reveals that the thermal stability of PLA-1%GO-Ag hybrids is higher than that of the PLA-Ag. From Fig. S3c, it is apparent that the residual weight of PLA-1%GO-Ag hybrid composites is higher than that of binary PLA-Ag composites and pure PLA. For neat PLA, the residual weight corresponds to the weight of carbonaceous residue after its complete decomposition to volatile gas compounds such as cyclic oligomers, lactide, acetaldehyde, carbon monoxide and carbon dioxide.84 For the PLA-1%GO-Ag hybrid nanocomposites, the residual weight is a combination 20 ACS Paragon Plus Environment

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GO, AgNP and carbonaceous residues after the complete degradation of composites. The amount of carbonaceous residue increases with increase of the AgNP loading. Table 2. TGA data of PLA and its nanocomposites. Samples

T10% (˚C)

T20% (˚C)

Tmax (˚C)

PLA

330.66

340.99

359.6

PLA-1%GO

341.53

350.31

365.4

PLA-1%Ag

332.47

342.99

361.6

PLA-3%Ag

334.31

346.45

365.4

PLA-7%Ag

329.91

344.48

364.1

PLA-1%GO-1%Ag

343.08

351.22

366.0

PLA-1%GO-3%Ag

342.16

351.52

362.8

PLA-1%GO-7%Ag

334.61

348.01

366.0

3.5. Silver ion release Figure 5 shows the Ag+ concentration released by the PLA-Ag and PLA-1%GO-Ag nanocomposite fiber systems immersed in distilled water from 1 to 28 days. PLA-1%Ag fibrous mat releases exceptionally small amount of silver ions upon immersion in distilled water. Increasing AgNPs content in the PLA-Ag composite to 3%, larger amounts of Ag+ ions released from fibrous mat can be detected. The silver ion release increases with increasing AgNPs content up to 7%. This is because elemental AgNPs of the PLA-Ag nanocomposites with higher filler contents can be oxidized electrochemically to silver ions by contacting with distilled water. Comparing with PLA-1%Ag nanocomposite, Ag+ ion concentration of 0.22 ppm and above can be detected in the PLA-1%GO-1%Ag counterpart by adding 1%GO upon immersion in water from 1 to 28 days. Moreover, Ag+ ion concentrations of the PLA-1%GO-3%Ag and PLA1%GO-7%Ag hybrid composites are much higher than those of PLA-3%Ag and PLA-7%Ag counterparts. This is due to the hydrophilic nature of GO containing hydroxyl and carboxylic 21 ACS Paragon Plus Environment


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groups. The Ag+ ion concentrations released from the hybrid composites increase with increasing immersion time as expected. From Fig. 5, PLA-1%GO-3%Ag, PLA-7%Ag and PLA-1%GO-7%Ag composite fibers release Ag+ ions markedly in D14. It is considered that Ag+ ion concentrations released from the PLA-Ag and PLA-GO-Ag composites depends greatly on the AgNP filler content and hydrophilicity of the composites. PLA-7%Ag sample with large Ag content releases high Ag+ ions as expected. Moreover, 7% AgNP filler addition can revert hydrophobic PLA to hydrophilic by reducing its contact angle as shown in the next paragraph, Fig. 6. Increasing hydrophilicity can also lead to an increase of released Ag+ ions from the PLA-7%Ag composite accordingly. As aforementioned, GO with oxygenated functional groups act as support layers to form AgNPs and to prevent AgNPs from segregation, thereby promoting uniform dispersion of AgNPs onto GO surface. This allows a more controlled release of Ag+ ions from the GO-Ag hybrid. In this respect, PLA-1%GO-3%Ag, and PLA-1%GO-7%Ag composite fibers exhibit high Ag+ ions in Day 14. Similarly, the additions of 1%GO-3%Ag and 1%GO-7%Ag to hydrophobic PLA reduces its contact angle markedly (Fig. 6). Therefore, the release of high Ag+ ion concentration from these samples facilitates bactericidal properties as expected.


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Figure 5. Plots of Ag+ release concentration vs immersion time of PLA-Ag and PLA-1%GO-Ag nanocomposite mats immersed in distilled water for different periods. PLA with a methyl side group generally exhibits hydrophobic behavior. Wettability of PLA and its nanocomposite fibrous mats can be determined by measuring the contact angles of water droplets on their fibrous mat surfaces. A lower value of contact angle demonstrates less hydrophobic nature of the fibers. Fig. 6 shows the appearances of contact angles with water of all samples studied. PLA fibrous mat exhibits a high contact angle of 131.57 ± 1.87°, demonstrating its hydrophobic nature. Adding 1%AgNPs to PLA decreases the contact angle slightly to 130.06 ± 0.94°, while 1%GO addition reduces to 126.86 ± 0.69° due to its oxygenated groups. However, the contact angle of the PLA-1%GO-1%Ag further reduces to 123.43 ± 0.74° by incorporating 1%GO. The PLA-1%GO-7%Ag nanocompsite fibrous mat exhibits the lowest contact angle of 102.34 ± 0.20°. Thus silver containing nanocomposites with lower contact angles favor the release of silver ions as described above.



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Figure 6. Appearances of water contact angles on PLA and its nanocomposite fibrous mats. 3.6. Antibacterial Activity Gram-negative E. coli usually predominates in hospital-acquired urinary tract and bloodstream infections in patients.59 Staphylococcus aureus is a major cause of bacteremia, causing higher morbidity and mortality, compared with bacteremia caused by other pathogens. There has been a significant rise in S. aureus bloodstream infection recently due to the increased frequency of joint replacements, and increased resistance of the bacteria to available antibiotics.85 Figure 7a shows the nutrient agar plate cultured with E. coli and then treated with AgNO3 (control), neat PLA, PLA-GO and PLA-Ag nanocomposites. The agar plate of E. coli treated with the PLAGO-Ag hybrid fibrous mats is shown in Fig. 7b. The control specimen (AgNO3) releases silver ions for antibacterial activity.63 Pure PLA shows no bactericidal activity as expected. Similarly, PLA-1%GO nanocomposite also shows little antibacterial activity. This implies that the addition of 1% GO to PLA does not increase its antibacterial property. From the little literature, conflicting results have been reported on the antibacterial activity of pure GO. Several 24 ACS Paragon Plus Environment

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researchers reported that GO sheets can cut bacterial membranes, encapsulate the bacteria for stopping their growth, and induce oxidative stress leading to the membrane disruption.86-88 Direct contact of the bacteria with extremely sharp edges of the GO leads to cell membrane damage and bacterial inactivation.86 However, Ruitz et al. reported that pure GO promotes bacterial growth and exhibits no bactericidal effect.89 Assuming standalone GO sheets can serve as nano-knives for cutting bacterial membranes, however, the incorporation of GO sheets into the PLA matrix in forming polymer nanocomposite leads to a loss of the cutting effect due to their binding with the PLA matrix. In this respect, sharp GO edges are covered with the polymeric material in the PLA-1%GO nanocomposite. Fig. 7a reveals that the PLA-1%Ag nanocomposite exhibits a small inhibition zone of 6.46 mm. This sample only releases exceptionally small amount of silver ions in contacting with water as mentioned previously. By increasing AgNP content to 3% and 7%, the inhibition zone increases largely to 11.06 and 11.18 mm. This is because PLA-Ag nanocomposite fibrous mats with high AgNP loadings can release larger amounts of Ag+ ions (Fig. 5). It is interesting to see that the inhibition zone against E. coli of PLA-1%GO-1%Ag hybrid is larger than that PLA-1%Ag nanocomposite (Figs. 7b and c). As aforementioned, GO with large surface area and many oxygenated groups serve as effective sites for anchoring AgNPs. As a result, E. coli bacteria trapped on the surface of electrospun nanocomposite mats are killed by the AgNPs of PLA-1%GO-1%Ag hybrid. So the amount of released Ag+ ions in this hybrid sample is sufficient to cause bacterial cell death. At 3% and 7% AgNPs loadings, the bactericidal effects of the PLA-1%GO-Ag hybrid system level-off. AgNPs have shown effective bactericidal activities against microorganisms. However, the antibacterial mechanisms of AgNPs are still open questions to researchers. Some mechanisms attribute to the AgNPs mediated membrane-damaging, while others consider the release of silver 25 ACS Paragon Plus Environment


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ions from nanoparticle surfaces. In the former case, excellent antibacterial effect of the AgNPs derives from their high surface area to volume ratio, which provides effective direct contact with microorganisms. Thus AgNPs attach to the cell membrane surface disturb its permeability, and penetrate cell membrane into the cytoplasm accordingly, thereby affecting intracellular processes and causing higher bactericidal activity.90,91 On the other hand, released silver ions tend to interact with the sulfur-containing compounds of bacteria. For example, Ag+ ions can interact with thiol groups of bacteria proteins, promoting the release of oxygen reactive species. This causes damage to proteins and DNA, affecting the DNA ability to replicate and resulting in final cell death.3,92 Recently, Bondarenko et al. indicated that a synergistic effect between these two mechanisms is required for bactericidal activity. Direct cell-nanoparticle contact facilitates the release of silver ions from AgNPs, thereby enhancing the amount of cellular uptake of particle associated Ag+ ions.93 Figure 8a shows the nutrient agar plate cultured with S. aureus and then treated with pure PLA, PLA-GO and PLA-Ag nanocomposites. The agar plate of S. aureus treated with PLA-GOAg hybrid fibrous mats is shown in Fig. 8b. The inhibition zone values for the PLA-Ag and PLA-1%GO-Ag nanocomposite fibrous mats are depicted in Fig. 8c. Apparently, the antibacterial inhibition zone values against S. aureus are smaller than those of the same samples against E. coli. These results clearly show that Gram-positive bacteria are less susceptible to Ag+ attack than Gram-negative bacteria. This is due to the structural difference in the composition of the cell walls of both bacteria strains. Gram-positive bacteria have a thick cell wall containing many peptidoglycan layers interspaced with teichoic acid. Teichoic acids are linear polymers consisting of polyol phosphates.94 The thick cell wall with peptidoglycan layers acts as a barrier for the penetration of Ag+ ions into the cytoplasm. By contrast, Gram-negative bacteria have a 26 ACS Paragon Plus Environment

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single layer of peptidoglycan surrounded by an outer membrane containing lipopolysaccharide. Also, teichoic acids are absent in the cell wall.95

Figure 7. Photographs of agar plates cultivated with E. coli and treated with (a) PLA, PLA1%GO, PLA-1%Ag, PLA-3%Ag, PLA-7% Ag and positive control samples, and (b) PLA1%GO-1%Ag, PLA-1%GO-3%Ag, PLA-1%GO-7%Ag hybrid fibrous mats. (c) Antibacterial inhibition zone values determined from (a) and (b) for the PLA-Ag and PLA-1%GO-Ag nanocomposite fibrous mats. 27 ACS Paragon Plus Environment


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Figure 8. Photographs of agar plates cultivated with S. aureus and treated with (a) PLA, PLA1%GO, PLA-1%Ag, PLA-3%Ag, PLA-7%Ag and positive control samples, and (b) PLA1%GO-1%Ag, PLA-1%GO-3%Ag, PLA-1%GO-7%Ag hybrid fibrous mats. (c) Antibacterial inhibition zone values determined from (a) and (b) for the PLA-Ag and PLA-1%GO-Ag nanocomposite fibrous mats. 3.7. Live/dead fluorescent imaging 28 ACS Paragon Plus Environment

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This assay is a fluorescent test employed for distinguishing live and dead bacteria. The bacteria cell is considered viable provide that the membrane is not damaged, while it is assumed dead if the membrane is disrupted or damaged. When used alone, SYTO 9 stains both live bacteria with intact membranes and dead bacteria with damaged membranes. However, propidium iodide (PI) penetrates only dead bacteria with damaged membranes, leading to a reduction in the SYTO 9 fluorescence when both dyes are used together. Therefore, live bacteria with intact cell membranes display fluorescent green since PI does not diffuse into these cells. As the cells are compromised, membrane porosity is developed, thus PI diffuses into the cell through compromised membrane and bind to DNA. As a result, dead bacteria display red fluorescence. Fig. 9 shows the live/dead fluorescent images of E. coli attached on pure PLA, PLA-1%GO, PLA-1%GO-1%Ag, PLA-1%GO-3%Ag and PLA-1%GO-7%Ag after 12 h incubation at 37 °C. The fluorescent images of S. aureus attached on these samples are shown in SI, Fig. S4. For pure PLA fibrous mat, both bacteria stain green color nearly, revealing the cells with intact membranes. Similar features are observed for the PLA-1%GO fibrous mat, with very few red spots appear in the fluorescent images, demonstrating that few bacteria destroy. Comparing with pure PLA and PLA-1%GO samples, PLA-GO-Ag hybrids inhibit bacterial adhesion and proliferation effectively, thereby preventing the formation of bacterial biofilm on their surfaces. In addition, the fluorescent images for both bacteria strains on binary PLA-Ag nanocomposites containing 1%, 3% and 7% AgNPs are presented in SI, Figs. S5 and S6. Comparing with binary PLA-Ag nanocomposites, PLA-GO-Ag hybrid fibrous mats exhibit lower cellular viability for both bacterial strains, indicating better antibacterial activity. Thus hybridization GO with AgNPs is effective in preventing bacteria colonization on the specimen surfaces of PLA-1%GO-Ag hybrid fibrous mats.



SYTO 9

Merge

PLA-1%GO-3%Ag

PLA-1%GO-1%Ag

PLA-1%GO

PLA

PI

PLA-1%GO-7%Ag

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Figure 9. Live/dead fluorescent images showing viability of E. coli on PLA, PLA-1%GO, PLA-1%GO-1%Ag, PLA-1%GO-3%Ag, PLA-1%GO-7%Ag samples. Cells are labelled with live (green) and dead (red) 3.8. Bacterial Morphologies By contacting electrospun fibrous mats with AgNPs fillers, bacterial cells undergo considerable morphological alterations as compared to pure PLA. Figs. 10a-c show representative SEM images of E. coli on the PLA, PLA-3%Ag and PLA-1%GO-3%Ag fibrous mats, while Figs. 10d-f are SEM images of S. aureus attached on the surfaces of these samples, respectively. E. coli on pure PLA exhibits typical rod shape feature. The results of the SEM micrographs reveal extensive damage to the cell structure of E. coli upon treatment with the PLA-3%Ag and PLA-1%GO-3%Ag fibrous mats, especially the latter sample. The cellular integrity of E. coli attached on the hybrid fibrous mat is lost completely. A large amount of cellular debris is observed around the cells demonstrating cell rupture. By contrast, S. aureus bacteria are more resistant against the attack of hybrid fibrous mat with GO-Ag fillers; the features of few bacteria remain unaffected by contacting with the GO-3%Ag fillers of the hybrid.



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Figure 10. SEM morphologies of E. coli on (a) PLA, (b) PLA-3%Ag and (c) PLA-1%GO3%Ag fibrous mat surfaces. SEM images of S. aureus on (d) PLA, (e) PLA-3%Ag and (f) PLA1%GO-3%Ag fibrous mat surfaces. 3.9. Quantitative antibacterial efficacy Figure 11a shows the percentage reduction in E. coli as a function of time for culture medium inoculated with neat PLA and its nanocomposite fibrous mats with AgNPs for 2 to 6h. PLA exhibits 0% bacterial reduction as expected. The PLA-1%Ag sample exhibits 12% bacterial reduction after 2h incubation and increases to 18% after 6h. The PLA-1%GO-1%Ag shows 29% bacterial reduction after 2h and further increases sharply to 85% after 6h. By increasing AgNPs contents to 3 and 7% for binary PLA-Ag and PLA-1%GO-Ag nanocomposite fibrous mats, a similar increasing trend in bacterial reduction is observed. The PLA-7%Ag and PLA-1%GO7%Ag hybrids show a slight higher bacterial reduction of 86% after 6h. This implies that we can reduce AgNPs content in the hybrid mats to 1% against E. coli in terms of economic consideration. Fig. 11b is a photograph of bacterial suspension inoculated with PLA and its 32 ACS Paragon Plus Environment

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nanocomposite fibrous mats with AgNPs fillers for 6h. The culture medium inoculated with PLA is cloudy, i.e. the medium is largely contaminated with E. coli. The addition of 1%Ag to PLA leads to a slight cloudy reduction in bacterial suspension. However, bacterial medium inoculated with PLA-1%GO-1%Ag becomes clearer due to a large bacterial reduction of 83% after 6 h incubation. This is because GO with large surface area can anchor more AgNPs onto its surface as discussed above. The same effect is observed for the suspensions inoculated with binary PLAAg and hybrid PLA-1%GO-Ag fibrous mats with higher AgNPs loading levels. SEM images reveal that the E. coli membranes are partially deformed after exposure to PLA-1%GO-3 %Ag for 2h (Fig. 11c). However, their cellular integrity is lost upon exposure for 6 h due to the rupture and a burst of membrane as indicated by the arrows, leading to the cytoplasm leakage (Fig. 11d). At 6h, bacterial cell debris are observed (Fig. 11e). On the other hand, a bacterial reduction of 82% after 6h can be achieved in the S. aureus suspension inoculated with PLA-1%GO-7%Ag hybrid containing largest AgNPs content (Fig. 12). Furthermore, the culture medium inoculated with PLA-1%GO-1%Ag hybrid only exhibits 22% after 6h incubation, while the suspensions with PLA-7%Ag and PLA-1%Go-3%Ag samples only reach 41% after 6h. Apparently, S. aureus with thick cell walls containing many peptidoglycan layers are resistant to the attack from lower AgNPs loadings of these samples. From Fig. 12, PLA-1%GO-7%Ag composite is more effective to kill S. aureus than PLA-7%Ag sample, although both composites contain the same Ag content. This is due to the presence of 1%GO in the PLA-1%GO-7%Ag composite. As aforementioned, GO serves as the anchor site for forming and dispersing AgNPs uniformly on its surface. A uniform dispersion of AgNPs enables the release of high Ag+ ions from the PLA-1%GO-7%Ag for killing bacteria (Fig. 5).



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Moreover, GO sheet provides a large surface area for bacterial attachment, thus allowing a more direct contact between the AgNPs and the surface of bacterial cells. The maximum bactericidal activity from quantification study was observed in S. aureus, for sample PLA-1%GO-7%Ag (Fig. 12). But the disk diffusion test shows lower zone of inhibition in S. aureus (Fig. 8c). The variation is due to the difference in sensitivity and nature of the tests. The test conditions, i.e. bacteria concentration and growth are different for these two measurements. Disk diffusion test employs a bacteria concentration of 1 x 105 CFU/ml and incubated at 37 °C for 18h, while turbidity test employs a bacteria concentration of 1 x 106 CFU/ml and incubated at 37 °C for 6h. As recognized, disk-diffusion assay is a qualitative screening method widely used for antimicrobial susceptibility testing.96 In contrast, turbidity test gives quantitative results by measuring the light absorbance of a bacteria culture medium or suspension. A bacterial culture acts as a colloidal suspension that blocks and reflects light passing through the culture. Bacteria absorb and scatter the light; the higher the bacteria concentration, the higher the turbidity. The absorbance (or optical density) is directly proportional to the bacteria concentration.


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Figure 11. (a) Percentage bacterial reduction vs time plots for E. coli culture medium inoculated with PLA and its nanocomposite fibrous mats with AgNPs fillers. (b) Photograph of E. coli culture suspensions inoculated with PLA and its nanocomposite fibrous mats with AgNPs fillers for 6h. SEM micrographs showing progressive loss of bacterial membrane integrity after (c) 2h, (d) 4h and (e) 6h incubation.



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Figure 12. Percentage bacterial reduction vs time plots for S. aureus culture medium inoculated with PLA and its nanocomposite fibrous mats with AgNPs fillers. 3.10. ROS generation Considering the cell membrane of E. coli is damaged by the Ag+ ions, thereby allowing subsequent interaction of silver ions with the thiol groups of bacteria proteins, facilitating the release of oxygen reactive species (ROS).3,92 As the Ag+ ions interact with thiol groups of bacteria, DNA loses its replication ability and the protein becomes inactivated.3 The inhibition of respiratory enzymes by AgNPs leads to the formation of ROS species such as hydroxyl radicals •

OH, superoxide ions O2•−, and H2O2, through oxidative decomposition of the cellular

components. This causes damage to proteins and DNA, resulting in cell death. Accordingly, oxidative stress is considered as an important factor of the DNA damage. In this study, oxidative stress in both bacteria strains after treatment with AgNPs nanofillers of polymer composite fibrous mats is determined by measuring intracellular ROS generation through the DCFH-DA assay. This DCFH-DA agent can diffuse through the cell membrane into the cell. Then it is 36 ACS Paragon Plus Environment

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deacetylated by the intracellular esterase to form non-fluorescent 2,7-dichlorofluorescein (DCFH). The DCFH reacts with ROS to give fluorescent 2,7-dichlorofluorescein (DCF).97,98 Fig. 13a shows the ROS level of E. coli in the presence of PLA, PLA-1%GO, PLA-(1-7) %Ag and PLA-1%GO-(1-7) %Ag specimens. Apparently, the PLA-based hybrids exhibit high ROS level, especially the PLA-1%GO-7%Ag amongst all the specimens studied. Moreover, PLA-(1-7) %Ag composites show lower ROS level than the PLA-based hybrids. Thus the ROS level is associated with the antibacterial activity of the composites containing AgNPs owing to the damaging effect to bacterial cell membranes by silver ions. This figure also reveals that the PLA-1%GO composite does not exhibit antibacterial effect. This is because GO fillers are embedded in the polymer matrix, so their sharp edges are sealed by the polymeric material. Thus the ROS level of E. coli due to PLA-1%GO composite is the same to that of control specimen, i.e. neat PLA. Fig. 13b is a schematic diagram illustrating the effect of AgNPs fillers of composite fibrous mats on the bactericidal activity of E. coli. First, fine AgNPs with large surface areas contact bacterial membrane. This is followed by releasing Ag+ ions and interacting with the thiol groups of bacteria proteins, promoting the generation of ROS. As a result, cytosolic components leak out of ruptured plasma membrane, which in turn results in cell death. Finally, the ROS level of S. aureus incubated with PLA, PLA-1%GO, PLA-(1-7) %Ag and PLA-1%GO-(1-7) %Ag specimens is shown in SI, Fig. S7. The PLA-based hybrids also exhibit high ROS levels comparing with PLA-(1-7) %Ag and PLA-1%GO specimens. In addition to bactericidal effect of AgNPs-based composite fibers as discussed above, the clinical use of AgNPs is a concern due to its toxicity. According to the literature, the toxic effects of AgNPs depends greatly on the dosage, size and shape of AgNPs, time point as well as contacted cell lines. For example, Hackenberg et al. reported reduced cell viability at a AgNP



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dose of 10 µg/mL (AgNPs < 50 nm, 1 h exposure) in human mesenchymal stem cells (MSCs).99 However, Samberg et al. found no toxicity for progenitor human adipose-derived stem cells up to 100 µg/mL (10 and 20 nm AgNPs for 24 h, and then differentiated for 14 days).100 Stoehl et al. indicated that spherical AgNPs (30 nm) are nontoxic towards alveolar epithelial cells (A549).101 Such variations in cell viability are due to different cellular binding and uptake of AgNPs. From these, the possible toxic effects of AgNP fillers of our composite fibers depends on the filler content and cell lines. It is suggested that AgNP fillers of composite fibers may induce toxic effects in MSCs, but show less toxicity towards human adipose cells and alveolar epithelial cells. Further work is needed to elucidate this issue.


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Figure 13. (a) Intracellular ROS generation of E. coli treated with PLA and its nanocomposite fibrous mats and (b) schematic diagram illustrating the release of Ag+ ions from AgNPs by contacting with bacterial cell membrane, followed by the ROS production and membrane rupture. 4. Conclusions In this article, we developed novel PLA-1%GO-Ag hybrid nanocomposite fibrous mats with bactericidal activity through electrospinning. For comparison, binary PLA-1%GO and PLA-Ag composite fibrous mats were also prepared. The bactericidal activities together with the morphological, tensile and thermal properties of these fibrous mats were investigated. Tensile measurements showed that the addition of 1%GO or 1-7% AgNPs to PLA improves its elastic modulus and tensile strength significantly. Further enhancements in tensile modulus and strength of PLA were achieved by hybridization GO with AgNP nanofillers. TGA tests revealed that the thermal stability of PLA is greatly improved by adding 1%GO and 1% Ag hybrid nanofillers. 39 ACS Paragon Plus Environment


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Agar disk diffusion results showed that PLA-1%GO nanocomposite had no inhibition zones against E. coli and S. aureus. Sharp edges of GO nanofillers were sealed by the PLA matrix, thus losing their cutting effect on both bacteria strains. However, GO nanofillers with a lateral width of micrometer scale provided effective anchoring sites for the AgNPs. The PLA-1%GO-(1-7) %Ag hybrid fibrous mats exhibited excellent antibacterial effect against E. coli on the basis of agar disk diffusion, live/dead fluorescent imaging, quantitative antibacterial efficacy and ROS measurements. Thus silver ions released from the hybrids upon contacting E. coli were sufficient to induce cell death by releasing ROS. By contrast, PLA-1%GO-(1-7) %Ag hybrid fibrous mat exhibited lower bactericidal effect towards S. aureus bacteria due to their thick cell walls. Quantitative antibacterial analysis revealed that the PLA-1%GO-7%Ag hybrid with the largest AgNPs loading had 82% inhibition for S. aureus after 6h incubation. Supporting Information TEM micrographs of PLA and its PLA-GO-Ag hybrid nanocomposite fibers are shown in Figure S1. Tensile stress-strain curves of electrospun PLA and its nanocomposite fibrous mats are shown in Figure S2. TGA curves and DTG thermograms of all samples studied are presented in Figure S3. Live/dead fluorescent images showing viability of S. aureus on all samples are shown in Figure S4. Fluorescent images for both E. coli and S. aureus on binary PLA-Ag nanocomposites containing 1%, 3% and 7% AgNPs are presented in Figures S5 and S6. Intracellular ROS generation of S. aureus treated with PLA and its nanocomposite fibrous mats are presented in Figure S7. Corresponding Author * E-mail address: [email protected] 40 ACS Paragon Plus Environment

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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Acknowledgements This study is supported by the Applied Research Grant (No. 9667126), City University of Hong Kong and Shenzhen Knowledge Innovation Program of Basic Research Items of Guangdong Province (JCYJ20140414090541811), China. References

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For Table of Contents Use Only: Development and Antibacterial Performance of Novel Polylactic AcidGraphene Oxide-Silver Nanoparticle Hybrid Nanocomposite Mats Prepared By Electrospinning Chen Liu,‡,† Jie Shen,‡,§ Kelvin Wai Kwok Yeung,§ and Sie Chin Tjong,*, † †

Department of Physics and Materials Science, City University of Hong Kong, Kowloon, Hong

Kong §

Department of Orthopedics and Traumatology, Li Ka Shing Faculty of Medicine, The

University of Hong Kong, Hong Kong


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Development and Antibacterial Performance of Novel Polylactic Acid

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